Understanding the molecular mechanisms underlying the human ?- to ?-globin gene switch has long been recognized as important in the treatment of sickle cell disease (SCD), since a wealth of evidence has demonstrated that increased fetal hemoglobin (HbF) significantly decreases the pathophysiology associated with this disease. Thus, knowledge of how to reactivate ?-globin (HbF) in adult erythropoiesis will benefit SCD patients. Our goal in this study is to understand ?-globin gene silencing during the adult stage of definitive erythropoiesis. Our clinical goal is to identify new molecular targets based on the outcome of this study that can be modulated therapeutically for up-regulation of ?-globin synthesis to treat SCD. Non-deletional hereditary persistence of fetal hemoglobin (HPFH) point mutations are likely to be highly informative regarding mechanisms of ?-globin gene repression and activation, but to date have not been studied extensively at the mechanistic level. The proposed study will test the hypothesis that HPFH mutations prevent silencing or maintain activation of ?-globin gene expression by abrogating recruitment of transcriptional repressor complex (repressosome) components normally located at the ?-globin gene or more distal intergenic regions of the locus, or alternately, by creating a favorable chromatin structure that allows the ?-globin genes to partly outcompete the ?-globin gene for interaction with the locus control region (LCR), or both. Several HPFH mutations have been introduced into our human ?-globin locus yeast artificial chromosome (?-YAC) including the -566, -195, -175, and -117 , A?-globin non-deletional HPFH point mutations and transgenic mice have been produced. In Specific Aim 1, we will study the mechanisms by which the - 566, -195, -175, and -117 HPFH mutations disrupt ?-globin gene repression and/or alter the ?-globin locus chromatin domain during development in vivo using murine models and molecular biology/biochemical approaches including transgene structure/expression studies, 3C, histone modification, DNase I sensitivity, co-activator/repressor recruitment, Bcl11A recruitment, and synergy between the HPFH mutations. In Specific Aim 2 we will examine how these four HPFH mutations alter DNA-binding protein complexes in the A?-globin promoter using proteomics of isolated chromatin segments (PICh) and Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) coupled with mass spectrometry (MS). In Specific Aim 3, we will generate and identify novel HPFH mutations within the A?-globin gene promoter using a cell-based reporter assay to select for HPFH mutations, followed by phenotypic characterization of these mutations in ?-YAC transgenic mice and determination of the DNA-binding activity at these sites. The knowledge we gain from these studies will reveal novel therapeutic targets for which highly-specific treatments may be developed to increase HbF for the treatment of SCD without the side-effects associated with broad-spectrum therapies.